Foods containing beneficial oral bacteria

ABSTRACT

Compositions and methods are described that provide controlled release of stabilized bacteria into the oral cavity on consumption of a food, candy, gum, or similar substance so as to promote colonization of oral surface by the bacteria. Bacteria are selected to treat an oral condition, such as oral disease, halitosis, and/or stained enamel on consumption of the food.

FIELD OF THE INVENTION

The field of the invention is compositions and methods for introducingprobiotic bacterial compositions to the mouth and/or gastrointestinaltract.

BACKGROUND

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Tooth surfaces are porous and can become stained or discolored by theuse of tobacco products, eating or drinking certain foods and beverages(e.g., coffee, tea and red wine), the buildup of dental plaque, theprocess of aging, diseases, trauma, medications, congenital conditions,and other environmental effects. Teeth are comprised of an inner dentinlayer, an outer enamel layer and an acquired pellicle. The acquiredpellicle is a proteinaceous layer derived from saliva that forms on thesurface of tooth enamel.

Extrinsic and intrinsic staining of the teeth can occur. Extrinsicstaining is staining of the acquired pellicle that can occur whencompounds such as tannins and polyphenolic compounds come in contactwith teeth during eating, drinking or smoking. These compounds thenbecome trapped in and tightly bound to the proteinaceous layer on thesurface of the teeth. Extrinsic staining can be removed by mechanicalmethods of tooth cleaning, such as brushing or flossing and by chemicalcleaning methods. Even with regular brushing and flossing, rapid or slowaccumulation can develop into noticeable intrinsic tooth discoloration.Intrinsic staining can be caused by staining compounds that penetratethe enamel layer and the dentin layer or can arise from sources withinthe tooth. Intrinsic staining is difficult to remove and cannottypically be removed by mechanical methods of tooth cleaning, but highchemical concentrations and/or prolonged chemical cleaning methods canbe used to remove some or all of this type of staining.

White, unstained teeth are considered cosmetically desirable. Teeth canbe whitened by, for example, mechanical cleaning methods, veneers thatare placed over the teeth, and chemical bleaching.

While tooth whitening products are known in the art, these products aretraditionally used by those seeking the cosmetic benefit of whiterteeth. However, there is a different population of consumers who desirewhiter teeth and/or more oral benefits including cleaner teeth,healthier gums, and decreased oral malodor. Therefore, there is a desireto provide oral hygiene products that can deliver oral care benefits inaddition to tooth whitening. Furthermore, oral hygiene time is typicallylimited and so there is a desire to deliver these oral care benefitsquickly and conveniently as part of a daily oral hygiene regimen.

U.S. Pat. No. 7,674,613, to Koga, describes foods that include a driedpreparation of Lactobacillus salivarius that has been selected tosuppress the growth of periodontopathic and cariogenic bacteria. Allpublications identified herein are incorporated by reference to the sameextent as if each individual publication or patent application werespecifically and individually indicated to be incorporated by reference.Where a definition or use of a term in an incorporated reference isinconsistent or contrary to the definition of that term provided herein,the definition of that term provided herein applies and the definitionof that term in the reference does not apply. Such an approach, however,is unlikely to be effective when applied to bacteria that are relativelylabile and/or moisture sensitive while in a lyophilized or dry state.This reduction in soluble protein concentrations reduces the productionof malodorous compounds in the mouth. United States Patent ApplicationPublication No. 2010/0047190, to Reindl et al, describes the use oflactic acid bacteria selected to bind to and aggregate Streptococcusnutans and that can be administered in a food, liquid, or film. Since noparticular measures are taken to protect the viability of thesebacteria, however, it is not clear if this approach can be successfulwith more labile species. This aggregation apparently reduces theincidence of dental caries. Since no particular measures are taken toprotect the viability of these bacteria, however, it is not clear ifthis approach can be successful with more labile species. United StatesPatent Application Publication No. 2015/0335577, to Agüeros Bazo et al,describes microencapsulation of probiotic bacteria mixtures using caseinand chitosan, which provides protection for the microencapsulatedbacteria until they reach the gut. United States Patent ApplicationPublication No. 2007/0098847, to Teissier, describes the use ofdehydrated and granulated “lactic bacteria” that has been coated with asolid vegetable fat in foods, which provides protection from moistureand release in the gut. Such methods, however, are not suitable fordelivery of labile bacteria species to the oral cavity.

Thus, there is still a need for convenient and palatable means forintroducing labile bacteria to the oral cavity.

SUMMARY OF THE INVENTION

The inventive subject matter provides compositions and methods in whichprobiotic bacteria are released into the oral cavity through consumptionof a foodstuff or application of a mucosal patch. Consumption of thefoodstuff or application of the mucosal patch provides release ofprobiotic bacteria for a time sufficient to at least transientlycolonize surfaces within the oral cavity, displacing potentially harmfulbacterial species.

One embodiment of the inventive concept is food substance (such as achewing gum, a hard candy, a wafer, a sprinkle, and/or a breath mint)that includes a stabilized bacterial formulation. The stabilizedbacterial formulation includes a stabilized non-pathogenic, hydrogenperoxide bacterial species or strain and a genetically modifiedLDH-deficient bacterial strain, and is provided in a solid or semi-solidconsumable vehicle. The stabilized bacterial formulation can includelyophilized bacteria and/or encapsulated bacteria. Such encapsulatedbacteria can include a matrix compound, for example casein, starch, achitosan, and/or an edible wax or oil. In such an embodiment the ediblewax or oil can have a melting point of greater than 35° C. Thestabilized bacterial formulation can include a stabilizer selected toimprove stability of the stabilized bacterial formulation. Suitablehydrogen peroxide-producing bacterial species or strains includeLactobacillus, Bifidobacteria, viridans Streptococcus, Leuconostoc,Pediococcus, and Lactococcus. Suitable genetically modifiedLDH-deficient bacterial strains include genetically modified strains ofStreptococcus nutans.

Another embodiment of the inventive concept is a mucosal patch thatincludes a support layer and a film layer. The film layer has a firstsurface that is coupled to the support layer, and includes a stabilizedbacterial formulation. The stabilized bacterial formulation includesisolated, non-pathogenic, hydrogen peroxide bacterial species or strainsand genetically modified LDH-deficient bacterial strains. Such bacterialstrains can be lyophilized and/or encapsulated (for example, within amatrix of casein, starch, a chitosan, and/or an edible wax or oil).Edible waxes or oils utilized can have a melting point in excess of 35°C. The support layer is sized to fit within a human oral cavity, and caninclude an adhesive. In some embodiments the mucosal patch also includesa barrier layer that is attached to a second surface of the film layerin a reversible fashion.

Another embodiment of the inventive concept is a method of colonizing anoral cavity with probiotic bacteria by providing a food substance (forexample, chewing gum, a hard candy, a wafer, and/or a breath mint) thatincludes a stabilized bacterial formulation. The stabilized bacterialformulation includes an isolated, non-pathogenic, hydrogen peroxidebacterial species or strain and a genetically modified LDH-deficientbacterial strain. Suitable hydrogen peroxide-producing bacterial speciesor strain include Lactobacillus, Bifidobacteria, viridans Streptococcus,Leuconostoc, Pediococcus, and Lactococcus. Suitable genetically modifiedLDH-deficient bacterial strain include genetically modified strains ofStreptococcus nutans. The food substance is retained within the oralcavity for at least 30 seconds. The probiotic bacteria can bestabilized, for example by drying (e.g. lyophilization) and/orencapsulation. Encapsulated preparation can include a matrix compound,such as casein, starch, a chitosan, and/or an edible wax or oil (forexample, and edible wax or oil with a melting point in excess of about35° C.). Instructions can be provided for consumption of the foodsubstance.

Another embodiment of the inventive concept is a method of at leasttransiently colonizing an oral cavity with probiotic bacteria byproviding a mucosal patch that includes a support layer, and alsoincludes a film layer comprising a first surface coupled to the supportlayer. The film layer includes a stabilized bacterial formulation, whichin turn includes isolated, non-pathogenic, hydrogen peroxide bacterialspecies or strains and genetically modified LDH-deficient bacterialstrains. The film layer is placed in contact with a mucosal surface ofthe oral cavity, and is retained in the oral cavity for a timesufficient to release at least a portion of the stabilized bacterialformulation into the oral cavity. The bacterial formulation canlyophilized and/or encapsulated bacteria. Such encapsulated bacteria caninclude a matrix compound such as casein, starch, a chitosan, and/or anedible wax or oil (or example, and edible wax or oil that melts attemperatures in excess of 35° C.). The mucosal patch can also include abarrier layer that is reversibly attached to a second surface of thefilm layer. In such embodiments the barrier layer is removed prior toplacing the film layer in contact with the mucosal surface.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingdrawing figures in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B schematically depict embodiments of the inventiveconcept in which a stabilized bacterial preparation is suspended withina rigid, protective shell. FIG. 1A shows such an embodiments in whichthe shell is essentially homogeneous. FIG. 1B shows such an embodimentin which the shell has inclusions that assist in breakdown in the oralcavity.

FIG. 2 schematically depicts an embodiment in which a stabilizedbacterial preparation is provided in a foam or gel.

FIG. 3 schematically depicts an embodiment in which a stabilizedbacterial preparation is provided in a homogeneous matrix.

DETAILED DESCRIPTION

Compositions and methods of the inventive concept provide protection ofa labile (e.g. moisture sensitive) bacterial preparation for deliveryinto the oral cavity using a packaging or matrix that providesprotection from water/humidity when intact but that releases readily inthe mouth when consumed as or as part of a foodstuff. Labile bacterialpreparations, such as lyophilized or otherwise dried bacterialpreparations that include a lactate dehydrogenase-deficient(LDH-deficient) mutans streptococcus, are thereby protected frommoisture prior to storage but are released into the oral cavity over aperiod of time when consumed. One should appreciate that compositionsand methods provide oral delivery of labile probiotic bacterialpreparations, under conditions which can provide for at least transientcolonization of surfaces within the oral cavity (such as teeth and/orgums). Such colonization can provide competition for bacteriaresponsible for dental caries and other oral conditions, and can providecompounds that improve the appearance of discolored teeth (such asperoxides).

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise. Also, as used in the descriptionherein, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise.

Unless the context dictates the contrary, all ranges set forth hereinshould be interpreted as being inclusive of their endpoints, andopen-ended ranges should be interpreted to include only commerciallypractical values. Similarly, all lists of values should be considered asinclusive of intermediate values unless the context indicates thecontrary.

The recitation of ranges of values herein is merely intended to serve asa shorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value with a range is incorporated into the specification asif it were individually recited herein. All methods described herein canbe performed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe invention and does not pose a limitation on the scope of theinvention otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

The following discussion provides many example embodiments of theinventive subject matter. Although each embodiment represents a singlecombination of inventive elements, the inventive subject matter isconsidered to include all possible combinations of the disclosedelements. Thus if one embodiment comprises elements A, B, and C, and asecond embodiment comprises elements B and D, then the inventive subjectmatter is also considered to include other remaining combinations of A,B, C, or D, even if not explicitly disclosed.

The instant invention provides methods and compositions that provide forat least transient colonization of the oral cavity by beneficialbacteria, which can act to displace oral bacteria that can cause variousunwanted oral conditions (for example, acid elaborating bacteria thatbreak down tooth enamel, etc.). Such displacement can providemaintenance or improvement of oral health, including, for example, toothwhitening. A composition of the invention comprises a stabilizedprobiotic bacterial composition that includes one or more isolated,non-pathogenic, hydrogen peroxide-producing species or strains ofbacteria, and optionally, LDH-deficient nutans Streptococcus. Certainbacteria can produce hydrogen peroxide at a level that can whiten teethas demonstrated herein for the first time. A composition of theinvention can influence or effect a desired change in appearance and/orstructure of a tooth surface. Examples of appearance and structuralchanges include, but are not necessarily limited to, whitening, stainbleaching, stain removal, plaque removal, and tartar removal.Furthermore, a composition of the invention can provide additional oralcare benefits such as displacement of bacteria that can cause dentalcaries, periodontitis, oral bacterial infections and diseases, oralwounds, Candida or fungal overgrowth, halitosis, or xerostomia-induceddental caries and associated periodontal diseases, or a combinationthereof to a subject.

Another embodiment of the inventive concept is a method for reducingstaining or discoloration of tooth surfaces using a compositioncomprising one or more non-pathogenic, hydrogen peroxide-producingviridans Streptococci species or strains, and/or one or morenon-pathogenic, hydrogen peroxide-producing Lactobacillus species orstrains and/or one or more non-pathogenic, hydrogen peroxide-producingBifidobacteria species or strains and/or one or more non-pathogenic,hydrogen peroxide producing Lactococcus species or strains and/or one ormore non-pathogenic, hydrogen peroxide producing Pediococcus species orstrains and/or one or more non-pathogenic, hydrogen peroxide producingLeuconostoc species or strains. In one embodiment of the invention thebacterial strains can be generally recognized as safe (GRAS), and cantransiently attach or adhere to a tooth surface by virtue ofelectrostatic interactions, van der Waals interactions, or protein orpolysaccharide adhesins on the bacterial surface that recognize andinteract with molecules present on the tooth surface. Examples ofviridans Streptococci species include, but are not limited to S.sanguis, S. parasanguis, S. gordonii, S. oralis, S. uberis, S. mitis, S.rattus, S. salivariaus, S. vestibularis, S. angionosus, S. constellatus,S. intermedius, S. mutans, S. sobrinus, S. milleri, S. cricetus, and S.mitior. Examples of Lactobacillus species include, but are not limitedto, L. acidophilus, L. jensenii, L. catenaforme, L. leichmanni, L.plantarum, L. johnsonii, L. gasseri, L. delbrueckii, L. casei, L.brevis, L. salivarius, L. gasseri, L. sobrius, L. rhamnosus, L. reuteri,L. fermentum, L. paracasei, L. dextranicum, and L. helveticus. Examplesof Bifidobacteria species include, but are not limited to B. angulatum,B. animalis, B. asteroides, B. bifidum, B. boum, B. breve, B.catenulatum, B. choerinum, B. coryneforme, B. cuniculi, B. dentium, B.gallicum, B. gallinarum, B. indicum, B. longum, B. magnum, B. merycicum,B. minimum, B. pseudocatenulatum, B. pseudolongum, B. psychraerophilum,B. pullorum, B. ruminantium, B. saeculare, B. scardovii, B. simiae, B.subtile, B. thermacidophilum, B. thermophilum, and B. urinali. Examplesof other non-pathogenic bacteria that can produce hydrogen peroxideinclude, without limitation, Pediococcus species, such as P.acidilactici, Leuconostoc species, such as L. mesenteroides, andLactococcus species such as L. lactis.

The quantity of hydrogen peroxide produced by bacteria can beexperimentally determined. See e.g. Hillman and Shivers, Arch. Oral.Biol., 33:395-401 (1988). The culture liquor of cells grown in thepresence of oxygen is incubated with 40 μg/ml horseradish peroxidase and0.4 μmol/ml o-dianisidine. After 2 minutes, the reaction is stopped bythe addition of 0.02 ml of 5N HCl. The optical density of the sample ismeasured at 410 nm and the hydrogen peroxide concentration of the sampleis calculated from a standard curve prepared using authentic hydrogenperoxide and an extinction coefficient at 230 nm of 81M⁻¹ cm⁻¹. In oneembodiment of the invention the bacteria can produce at least about 0.5,1, 2, 5 mM or more of H₂O₂ or any range or value between about 0.1 andabout 5 mM.

In one embodiment of the invention a composition of the inventioncomprises one or more isolated Streptococcus oralis strains and/or oneor more S. uberis strains. Compositions of the invention can optionallycomprise one or more isolated strains or species of mutans streptococcusthat are LDH-deficient. The combination of non-pathogenic, hydrogenperoxide-producing bacteria and/or mutans streptococcus provides asignificant practical advantage in that the combination can used toreduce oral populations of organisms responsible for dental caries,periodontitis, oral bacterial infections and diseases, oral wounds,Candida or fungal overgrowth, halitosis, or xerostomia-induced dentalcaries or periodontal disease, while also whitening the teeth.

Streptococcus oralis (previously known as S. sanguis Type II) and S.uberis are important components in maintaining the normal, healthybalance of microorganisms that comprise the periodontal flora. SeeSocransky et al., Oral Microbiol. Immunol. 3:1-7 (1988); Hillman andShivers, Arch. Oral. Biol., 33:395-401 (1988); Hillman et al., Arch.Oral. Biol., 30:791-795 (1985). S. oralis can also be found in dentalplaque and has been demonstrated to correlate with periodontal health,in particular by interfering with the colonization by periodontalpathogens such as Aggregetobacter actinomycetemcomitans, Porphyromonasgingivalis, Peptostreptococcus micros, and Campylobacter rectus.Compositions of the invention can comprise one or more isolated strainsof S. oralis, for example, ATCC 35037, ATCC 55229, ATCC 700233, ATCC700234 and ATCC 9811. Other strains of S. oralis include KJ3 and KJ3sm.KJ3sm is a naturally occurring genetic variant of KJ3 that is resistantto 1 mg/ml streptomycin. The streptomycin resistance is advantageousbecause it provides a marker for easy isolation of the bacteria.Additionally, streptomycin resistant strains are slightly attenuated anddo not survive as long in an oral cavity as wild-type strains. Thisproperty is useful where the goal is to non-persistently colonize theoral cavity of an animal with the bacteria.

S. uberis can also be found in dental plaque and has been demonstratedto correlate with periodontal health, in particular by interfering withthe colonization by periodontal pathogens such as Tannerellaforsythensis, P. micros, C. rectus, and Prevotella melaninogenica.Compositions of the invention can comprise one or more isolated strainsof S. uberis, for example, ATCC 13386, ATCC 13387, ATCC 19435, ATCC27958, ATCC 35648, ATCC 700407, ATCC 9927, strain KJ2 or strain KJ2sm.KJ2sm is a naturally occurring genetic variant of KJ2 that is resistantto 1 mg/ml streptomycin and provides the same advantages as forstreptomycin-resistant strains of S. oralis. One or more isolatedstrains of S. oralis or one or more isolated strains of S. uberis, orboth, can be used in compositions and methods of the invention.

Compositions of the invention can comprise one or more isolated mutansstreptococcus bacteria species deficient in the production of lacticacid. These species include, for example, S. rattus, S. cricetus, S.mutans, S. sobrinus, S. downeii, S. macacae, and S. ferus. A mutansstreptococcus of the invention does not substantially produce L(+)lactate dehydrogenase (LDH). Such a strain is termed an LDH-deficientstrain. An LDH-deficient strain of mutans streptococcus produces 25%,20%, 10%, 5%, 2%, 1%, or less than 1% of the lactic acid produced bywild-type strains of mutans streptococcus under similar conditions. AnLDH-deficient mutans streptococcus strain can be a naturally occurringstrain of mutans streptococcus or a genetically modified strain ofmutans streptococcus. LDH-deficient mutans streptococcus can competewith and/or displace pathogenic bacteria such as S. mutans, a principaletiological agent of dental caries, in the oral cavity. LDH-deficientmutans streptococcus stains will compete with S. mutans for the samenutrients, colonization sites, etc. Therefore, LDH-deficient mutansstreptococcus strains can be used to, for example, prevent and/or treatdental caries. LDH-deficient strains of mutans streptococcus arenon-pathogenic, alter the microenvironment of the oral cavity to preventcolonization or outgrowth of pathogenic organisms, and/or displacepathogenic organisms from the oral cavity where the pathogen is part ofthe host's indigenous flora.

Examples of LDH-deficient mutans streptococcus strains include, forexample, S. rattus JH145 (ATCC 31377) (a spontaneous,naturally-occurring LDH-deficient mutant) and JH140 (ATCC 31341) (achemically-modified LDH-deficient mutant). See e.g., Stanshenko &Hillman, Microflora of plaque in rats following infection with anLDH-deficient mutant of Streptococcus rattus, Caries Res. 23:375-377(1989); Hillman, Lactate dehydrogenase mutants of Streptococcus mutans:Isolation and preliminary characterization. Infect. Immun. 21:206-212(1978); see also Abhyankar et al., Serotype c Streptococcus mutansmutatable to lactate dehydrogenase deficiency. J. Dent. Res. 64:1267-71(1985).

An LDH-deficient strain of mutans streptococcus can be derived from amutans streptococcus strain using, for example, chemical or physicalmutagenesis techniques. Strains that are mutagenized using thesetechniques are considered genetically modified strains. For example, amutans streptococcus strain can be subjected to mutagens such as nitrousacid, formic acid, sodium bisulphate, UV light, base analog mutagens,including for example, 5-bromo-deoxyuridine (5BU), alkylators such asethyl methane sulfonate (EMS), methyl methane sulfonate (MMS),diethylsulfate (DES), and nitrosoguanidine (NTG, NG, MNNG). See e.g., InVitro Mutagenesis Protocols, Braman, Ed., Humana Press, 2002.

Naturally-occurring, spontaneous LDH-deficient mutans streptococcusstrains can be prepared using methods disclosed in, for example,Hillman, Lactate dehydrogenase mutants of Streptococcus mutans:isolation and preliminary characterization. Infect. Immun. 21:206-212(1978). Spontaneous LDH-deficient mutants occur at the rate ofapproximately 10⁻⁵ frequency. See Johnson et al., Cariogenic potentialin vitro in man and in vivo in the rat of lactate dehydrogenase mutantsof Streptococcus mutans. Arch. Oral Biol. 25:707-713 (1980).

Naturally-occurring, spontaneous LDH-deficient strains of mutansstreptococcus can be differentiated from LDH-producing strains of mutansstreptococcus by plating the bacteria on glucose tetrazolium medium.LDH-deficient mutans streptococcus colonies will be bright red andrelatively larger in size than colonies of the parent strain, which arewhite and relatively smaller in size on the glucose tetrazolium medium.Naturally-occurring, spontaneous LDH-deficient strains of mutansstreptococcus can be used in a composition of the invention.

An LDH-deficient strain of S. rattus has been isolated. Briefly, aculture of S. rattus BHT-2 was grown overnight to saturation in ToddHewitt broth, and diluted samples were spread on glucose tetrazoliummedium to give approximately 300 colonies per plate. Wild-type, acidproducing colonies are white on this medium. LDH-deficient mutants arebright red. S. rattus JH145 was one red colony amid approximately100,000 white colonies that were screened. S. rattus JH145 is thereforea naturally-occurring, LDH-deficient mutant.

LDH-deficient strains of mutans streptococcus, such as LDH-deficientmutants of S. rattus BHT-2, produce less total titratable acid whenincubated in the presence of glucose and other sugars or polyols, makesubstantially less lactic acid when incubated in the presence of glucosein the case of resting and growing cultures, adhere better tohydroxyapatite and accumulate more plaque when grown in the presence ofsucrose. LDH activity can be assayed as described by Brown &Wittenberger (J. Bacteriol. 110:604, 1972).

Terminal pH can be determined by subculturing strains (1:100) inTodd-Hewitt broth containing 1% glucose. After 48 hours incubation incandle jars at 370 C, the absorbance at 580 nm and pH of the culturescan be determined. Lactic acid concentration of cultures can bedetermined by gas-liquid chromatography. See Salanitro & Muirhead,Quantitative method for the gas chromatographic analysis of short-chainmonocarboxylic and dicarboxylic acids in fermentation media. Appl.Microbiol. 29:374-381 (1975); Hillman et al., Acetoin production bywild-type strains and a lactate dehydrogenase-deficient mutant ofStreptococcus mutans. Infect. Immun. 55:1399-1402 (1987). Additionally,any genetic modification techniques known to those of skill in the artcan be used to create an LDH-deficient mutans streptococcus strain froman LDH-producing mutans streptococcus parent strain. For example, an LDHgene or a portion of an LDH gene can be deleted or mutagenized,including, for example, insertional mutagenesis techniques. Othermutagenesis techniques include, for example, homologous recombination,recursive sequence recombination, oligonucleotide-directed mutagenesis,site-directed mutagenesis, error-prone PCR, phosphothioate-modified DNAmutagenesis, uracil-containing template mutagenesis, gapped duplexmutagenesis, point mismatch repair mutagenesis, repair-deficient hoststrain mutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,site saturation mutagenesis, ensemble mutagenesis, recursive ensemblemutagenesis, and chimeric nucleic acid creation. Therefore, any geneticmodification technique that disables an LDH gene can be used to producean LDH-deficient mutans streptococcus strain. In one embodiment of theinvention, the LDH-deficient strains, whether naturally-occurring orgenetically-modified mutants, have a reversion frequency less than 10⁻⁷and produce less than about 10% of the parental level of lactatedehydrogenase activity.

The use of two or more different species of bacteria can provide anadvantage over using a single species. This is because different speciesof bacteria colonize different surfaces or portions of teeth. Therefore,the use of more than one species of bacteria can be used to occupy allor most surfaces of the teeth, whereas the use of only one species ofbacteria may result in certain surfaces or portions of the teeth beinguncolonized. Therefore, all surfaces of the teeth are exposed towhitening action.

Compositions of the invention can further comprise one or more carbonsources that are metabolizable by the one or more isolated,non-pathogenic, hydrogen peroxide-producing bacterial species or strainsor the one or more lactate dehydrogenase deficient mutans Streptococcusspecies or strains or both types of species or strains. Carbons sourcesinclude, but are not limited to, for example, glucose, sorbitol,mannitol, fructose, galactose, maltose, sucrose, xylose, lactose,glycerol or combinations thereof.

The compositions of the invention can include a pharmaceuticallyacceptable or nutritionally acceptable carrier. The carrier isphysiologically compatible with the oral cavity of the subject to whichit is administered. Carriers can be solid-based, dry or at leastpartially low water activity materials suitable for formulation intotablet, capsule, lozenge, and/or powdered form. A carrier can also be aliquid or gel-based materials, for formulations into liquid, gel, and/orchewing gum forms. Suitable liquid or gel-based carriers include but arenot limited to: water, physiological salt solutions, urea, alcohols andderivatives, and glycols (e.g. ethylene glycol or propylene glycol). Thecomposition of the carrier can be varied so long as it does notinterfere significantly with the therapeutic activity of the bacterialstrains of the invention.

In other embodiments the carrier can be in the form of an oral patch.Such an oral patch can include stabilized beneficial bacteria and anadhering surface that at least temporarily attaches to a surface of theoral cavity (for example the hard palate, soft palate, gums, and/ortooth surfaces) and maintains the position of the patch within the oralcavity following application and a protective surface. The protectivesurface can be at least partially permeable to saliva, and serves torelease the stabilized bacteria over time following application. An oralpatch of the inventive concept can release stabilized beneficialbacteria for from 1 minute to 20 minutes or more following application,thereby supporting colonization of the oral cavity by the releasedbacteria rather than simple transfer to the lower digestive tract. Forexample, in some embodiments the protective surface can be porous, wherethe pores are dimensioned to release bacteria into the oral cavity. Suchpores can range in size from greater than 0.02 μm in diameter to up toabout 2 mm in diameter. In such an embodiment the patch can be providedwith pores that are occupied by a substance that dissolves in the oralcavity, such as a water soluble material or a starch-based material thatis broken down by salivary amylase.

In some embodiments such a patch is applied to a surface of the oralcavity and allowed to adhere, then left in place for a prescribed periodof time (for example, about 10 seconds to about 20 minutes) before beingremoved. In other embodiments the protective surface can be dissolvablewithin the oral cavity, for example by being at least partially watersoluble and/or including a starch component degradable by oral amylase.In such an embodiment the protective surface can be formulated to remainat least partially intact through a prescribed treatment period (forexample, from about 10 seconds to about 20 minutes). In some embodimentsthe patch can include an adhesive that aids in adherence to a surface ofthe oral cavity. In other embodiments the patch can not include such anadhesive, and rely on taction to remain in place.

In some embodiments a composition of the inventive concept can beformulated to be suitable for oral administration as a consumablesubstance in a variety of ways, for example in a solid, semi-solid,liquid (including, e.g. a viscous liquid, a paste, a gel, or asolution), a dried mass, a dentifrice, a mouth wash, an oral rinse, aliquid suspension, a topical agent, a powdered food supplement, a paste,a gel, a solid food, an oral rinse, a packaged food, a wafer, lozenge,chewing gum and the like. Other formulations will be readily apparent toone skilled in the art. A composition of the invention can include anutrient supplement component and can include any of a variety ofnutritional agents, as are well known, including vitamins, minerals,essential and non-essential amino acids, carbohydrates, lipids,foodstuffs, dietary supplements, and the like.

In a preferred embodiment of the inventive concept the composition isprovided as a food, which is placed in the mouth and chewed for a periodof time prior to swallowing. Such chewing can serve to expose thestabilized bacterial preparation of the composition and rehydrate it,while distributing it throughout the oral cavity for a period of timesufficient to establish at least transient colonization by the bacteria.This can be accomplished by using a single portion that is retained inthe mouth (for example, a chewing gum) and/or by providing multiple“bites” that are consumed over time (for example, a food or candy bar).Suitable times for establishing at least transient colonization of theoral cavity are for about 30 seconds, about 45 seconds, about 1 minute,about 90 seconds, about 2 minutes, about 3 minutes, about 4 minutes,about 5 minutes, or more than about 5 minutes. Such times can be a timethrough which a single portion is held in the mouth, or can be asummation of times during which different portions are held in the mouthduring consumption.

Compositions of the invention can also include natural or syntheticflavorings and food-quality coloring agents, all of which are compatiblewith maintaining viability of the bacterial species or strains of theinvention.

A composition of the invention can include one or more gelling agentsthat can act as an adhesive agent to adhere the composition to theteeth. The concentration of the gelling agent may be greater than about2, 4, 6, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80 or less than about 80,70, 60, 50, 40, 30, or 20 percent by weight of the composition.

Suitable gelling agents and adhesion agents useful in the presentinvention include, for example, silicone, polyethylene oxide, polyvinylalcohol, poly alkyl vinyl ether-maleic acid copolymer (PVM/MA copolymer)such as, Gantrez A N 119, AN 139, and S-97, polyvinyl alcohol,polyacrylic acid, Poloxamer 407 (Pluronic), polyvinyl pyrrolidone-vinylacetate copolymer (PVP/VA copolymer), such as Luviskol V A, and PlasdoneS PVP/VA, polyvinyl pyrrolidone (PVP, e.g., K-15 to K-120),Polyquaterium-11 (Gafquat 755N), Polyquaterium-39 (Merquat plus 3330),carbomer or carboxypolymethylene (Carbopol), hydroxy propyl methylcellulose, hydroxy ethyl cellulose, hydroxy propyl cellulose, cornstarch, carboxymethyl cellulose, gelatin and alginate salt such assodium alginate, natural gums such as gum karaya, xanthan gum, Guar gum,gum arabic, gum tragacanth, and mixtures thereof.

A humectant or plasticizer can be present in compositions of theinvention. Humectants or plasticizers include, for example, glycerin,glycerol, sorbitol, polyethylene glycol, propylene glycol, and otheredible polyhydric alcohols. The humectants or plasticizers can bepresent between at about 1% to about 99%, about 10% to about 95%, or atbetween about 50% and about 80% (or any range between 1% and 99%) byweight of a composition.

Bacteria of the invention can be prepared for use in a fermentor orsimilar culture vessel. The bacteria can be harvested from the fermenterand further processed (for example, concentrated) for stability or aspart of a process that confers stability under storage conditions.Bacterial preparations can be considered stable if they providesufficient viable bacteria for their intended use after at least 6months of storage at ambient or reduced (for example, down to 2-8° C.,0° C., or −20° C.). Bacteria of the invention can be stabilized bydrying, for example, dehydration, lyophilization, and/or spray drying.Spray drying generally comprises spraying a suspension of bacteria in avessel and under a steam of hot air. Such drying can be performed in thepresence of compounds that improve viability following drying, such astrehalose and/or other stabilizing carbohydrates, serum albumin,ovalbumin, etc. As noted above, such dried bacterial preparations can befurther protected by suspension in a water-immiscible fluid, such as anedible oil. Bacteria can also be stabilized for use bymicroencapsulation (see e.g. U.S. Pat. No. 6,251,478), freeze-drying, orby coating with a protective substance such as, for example, lipidmaterial such as triacylglycerols, waxes, organic esters, soybean oil,cottonseed oil, palm kernel oil, and esters of long-chain fatty acidsand alcohols.

The invention provides methods for delivering a composition thatprovides probiotic bacteria that can at least transiently colonize theoral cavity, by the providing a food substance that includes astabilized form of the probiotic bacteria. Consumption of the foodsubstance provides a bolus of the probiotic bacteria, and does so over aperiod of time (e.g. from 10 seconds to 30 minutes) the permits saidbacteria to adhere to one or more surfaces of the oral cavity of theconsumer. In competing for other bacteria within this limitedenvironment one or more oral care benefits can be realized. In someembodiments one or more bacterial strains within the probiotic bacteriapopulation can elaborate compounds (such as peroxides) that provideadditional benefits, including a reduction in discoloration in toothenamel.

The bacterial species or strains can be present in a composition of theinvention in an amount effective to provide sufficient viable bacteriafor at least transient colonization of the oral cavity. As noted above,displacement of existing bacterial population within the mouth bybacteria of the probiotic bacteria mixture can reduce, prevent and/orameliorate unwanted effects that resulted from the presence of theexisting bacteria population. Such unwanted effects include symptoms ofdental caries, periodontitis, bacterial infections or diseases, oralwounds, Candida or fungal overgrowth, halitosis, or xerostomia-induceddental caries or periodontal disease or to alleviate, reduce, prevent,or ameliorate stains or discoloration on the teeth either permanently ortemporarily.

An effective amount is an amount of a composition of the invention thatis sufficient to provide the desired colonization, but low enough toavoid serious negative effects (at a reasonable benefit/risk ratio). Theeffective amount of a composition of the invention may vary with theparticular native bacteria population present, with negative effectspresent in the oral cavity due to the presence of this native bacteriapopulation, the age and physical condition of the patient being treated,the duration of over which the probiotic bacteria will be used, thenature of any concurrent medical interventions, the specific form of thefood source employed, and the particular vehicle from which theprobiotic bacteria composition is applied.

The VITA 3D Master™ Shade Guide provides a range of tooth shades thatvaries from very light to very dark. A total of 29 tooth shadesconstitute the entire range of colors between these two endpoints on ascale of brightness. In one embodiment of the invention, methods of theinvention provide a change of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15 or more (or any value or range in between 1 and 29) VITA3D Master™ shades of brightness.

The inventive concept provides compositions and methods in which apreparation of dried, lyophilized, or otherwise bacteria useful in aprobiotic mixture are introduced to the oral cavity through the use of afood or similar solid or semi-solid consumable. Such bacteria caninclude lactate dehydrogenase-deficient mutans streptococcus strains,for example mutans streptococcus that has been exposed to mutagens orgenetically engineered to show reduced or absent lactate dehydrogenaseactivity. Alternatively or in addition, such bacteria can includebacterial strains that generate hydrogen peroxide in the oral cavity. Asopposed to protecting such compositions for release in the gut (e.g.stomach, small intestine, large intestine), compositions and methods ofthe inventive concept provide protection and stabilization of such driedor lyophilized bacterial preparation during storage, and release asignificant portion of the dried or lyophilized bacterial preparationwhile in the oral cavity (preferably over a period of time of up to 15minutes). For example, up to about 25%, 30%, 35%, 40%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater than about 95% of thetotal bacterial content of a composition of the inventive concept can beexposed to the interior of the oral cavity of a user during normalconsumption (e.g. excluding circumstances where a food substanceintended for chewing or sucking is immediately swallowed intact).

Embodiments of the inventive concept can include bacterial preparationsthat are useful in colonizing an oral cavity and displacing potentiallyharmful native bacteria, but whose use is hampered by a relative lack ofstability and/or viability upon storage under conventional conditions(i.e. labile bacteria). In some embodiments simple drying orlyophilization does not provide suitable stability (for example, for atleast 12 months) for labile bacteria under storage conditions. Suitablestorage conditions include storage at 2° C. to 8° C. (e.g. conventionalrefrigeration), storage at 15° C. to 25° C. (e.g. “room” temperature)under low to near-saturating humidity conditions. In some embodimentsadditional packaging is provided in the form of a wrapper or othersealed enclosure that surrounds the foodstuff. Such a wrapper or sealedenclosure can provide a moisture barrier, and in some embodiments canadditionally enclose a dessicating agent (e.g. silica gel). In someembodiments such a wrapper or sealed enclosure can be sparged orotherwise filled with a non-reactive gas (e.g. N₂, Ar, CO₂) prior tosealing. While such a wrapper or sealed enclosure can provide protectionof the labile bacterial preparation, preferred embodiments of theinventive concept can include additional protective features, such ascoating, encapsulations, etc. These can be useful in extending theuseful life of the labile bacterial preparation, particularly onextended storage or on partial consumption of the foodstuff. In someembodiments such additional protective features can provide sufficientprotection that a wrapper or sealed enclosure is not necessary.

In some embodiments of the inventive concept the labile bacterialpreparation can be provided as a dried and/or lyophilized preparation,for example as a powder or granules. In such an embodimentlyophilization or drying can be performed in the presence of stabilizingagents that act to extend the useful lifetime of the labile driedbacteria, thereby incorporating such stabilizing agents into the powderor granules. In other embodiments such stabilizing agents can beprovided in or as part of a matrix, in which the dried or lyophilizedbacterial preparation is imbedded or suspended in the form of particlesof powder, individual granules, etc. Suitable stabilizers includeproteins (e.g. albumin, mammalian gelatin, teleost gelatin, milk solids,serum products, etc.), free radical inhibitors, reducing agents, sugarsor sugar polymers (e.g. glucose, trehalose, sucrose, sorbitol, mannitol,dextran, cyclodextrin, etc.), amino acids (e.g. glycine), surfactants(e.g. polysorbate 20, polysorbate 80) and/or synthetic polymers (e.g.polyvinylpyrrolidone).

In some embodiments, as shown in FIG. 1A, a food composition includes amatrix 100 having one or more stabilized bacterial inclusion(s) 110, andcan be provided with a protective shell or capsule 120. In someembodiments such a protective shell can be dimensioned (e.g. 1 mm to 20mm in a given linear dimension) and have a thickness (e.g. 0.05 mm to 2mm) that permits the shell 120 to be ruptured during normal chewing. Forexample, such an embodiment could be provided in the form of edible“sprinkles”, such as are used in food decoration, or as a candy orlozenge (or a component of same). In some embodiments the protectiveshell or capsule 120 can be composed of a material that offers moistureresistance but that is degraded by or soluble in substances found in themouth (such as saliva or components thereof). For example, such a shellcan be at least partially water soluble, and/or include a starch orstarch-polymer component that is reactive with the amylase found insaliva. In such an embodiment the shell or capsule 120 would provideprotection until entered into the oral environment, then degrade andrelease the stabilized bacteria 110.

In some embodiments, as shown in FIG. 1B, such a shell 120 canincorporate one or more reactive inclusion(s) 130 that are stable understorage conditions but that disrupt the integrity when of the shell 120when exposed to the oral environment. Such reactive inclusions 130 can,for example, rapidly dissolve (e.g. less than 5 minutes) in the oralenvironment, thereby weakening the shell 120 and exposing the interiorcontents. Alternatively, such reactive inclusion 130 can react withcomponents of the oral cavity to generate gases that pressurize anddisrupt the shell 120. Suitable reactive inclusion 130 include, forexample, mixtures of an edible organic acid (e.g. ascorbic acid, malicacid, etc.) and a carbonate or bicarbonate salt (e.g. CaCO₃, Na₂CO₃,NaHCO₃, etc.). Exposure of such a mixture to aqueous solutions (such assaliva) can result in dissolution of the reactive inclusion and rapidgeneration of CO₂ gas. The resulting loss in the integrity of the shell120 can lead to release of the stabilized bacteria 110 into the oralcavity over time (e.g. from 1 second to 10 minutes).

In other embodiments, as shown in FIG. 2, inclusions of stabilizedbacteria 110 are provided in a stiff (i.e. non-conforming to acontainer's shape) low water activity, disruptable foam or gel 200 thatis loses structural integrity within the oral cavity. In someembodiments such a foam, gel, and/or aerogel 200 can includestabilizers, coloring agents, and/or flavorants. Loss of structuralintegrity can be due to factors such as solubility and/or degradation ofone or more components of the foam. In some embodiments loss ofstructural integrity can be provided by mechanical disruption within themouth, for example by chewing. Such a stiff foam, stiff gel, and/or dryfoam (e.g. aerogel) carrying inclusions of dried or lyophilized bacteriacan be produced in bulk and then trimmed to a desirable size and/orshape, such as a size and shape suitable for inclusion in a breakfastcereal, inclusion in food bar, as a chewing gum, etc. In such anembodiment stabilized bacteria near the periphery may be moresusceptible to environmental factors while those towards the centerremain protected. Suitable components for such a disruptable foam, gel,and/or aerogel include edible starches, edible proteins (such asgelatin), and/or edible gums (such as xanthan gum). In some embodiments,the foam, gel, and/or aerogel can be produced as a wet foam or gel thatincludes probiotic bacteria suitable for at least transiently colonizingthe oral cavity and dried or lyophilized to provide a low moisture,disruptable material and a lyophilized or dried bacterial preparation inthe same process step.

In another embodiment of the inventive concept, as shown in FIG. 3,inclusions of dried or lyophilized bacteria 110 are provided in alow-melting point (e.g. less than or equal to about 45° C.) matrix 300.For example, such a matrix can have a melting point that is within 5° C.of body temperature within an oral cavity. Introduction into the oralcavity results in melting of the low-melting point matrix, which in turnreleases the stabilized bacteria inclusions 110 into the oral cavity. Ina preferred embodiment, the low-melting point matrix is selected to havea melting point temperature that is tolerated by the dried orlyophilized bacteria for from 5 minutes to 1 hour, thereby permittingformulation through simple addition of the dried or lyophilized bacteriato the melted low-melting point matrix. Suitable low-melting pointmatrices include edible oils and/or waxes, such as dairy butter, lard,margarine, mutton tallow, coconut oil, cocoa butter, milk fat, palmkernel oil, and/or palm oil. Such a low-melting point matrix carryinginclusions of stabilized bacteria can, for example, form at least partof a food bar, decorative frosting, pastry filling, and/or confection.

In some embodiments stabilized bacteria can be provided as a suspensionin an edible oil that is liquid at ambient temperature, or that becomesliquid as it approaches body temperature (i.e. 37° C.). Such an oilsuspension can be provided as a liquid, gel, and/or paste inclusion(e.g. creamy filling) of a food or confection, such that it issurrounded by a conventional foodstuff. For example, such an oilsuspension can be provided as a liquid center of a gum, lozenge, orcandy, such that the bacterial suspension is released into the mouth onchewing. Similarly, such a gel or paste can be incorporated into afilling of a pastry or similar foodstuff. In a preferred embodiment suchan oil suspension can include a flavorant (e.g. a sweetener or otherflavor). Suitable edible oils include coconut oil, corn oil, cottonseedoil, olive oil, palm oil, peanut oil, canola/rapeseed oil, saffloweroil, sesame oil, soybean oil, and/or sunflower oil.

One embodiment of the invention provides a method of non-persistentlycolonizing an oral cavity of a subject with therapeutically-effectivebacteria comprising providing a subject with a food item as describedabove, wherein the subject exposes the food item to their oral cavity byconsumption. In one embodiment of the invention the administeredbacterial strains do not permanently colonize the oral cavity, ratherthe strains are present in the oral cavity for about 1 day, about 1week, about 2 weeks, about 3 weeks, about 1 month, about 3 months orabout 12 months after administration of the bacteria.

Compositions of the invention can be administered at a dose of about1×10³, 1×10⁵, 1×10⁷, 1×10⁹, or 1×10¹¹ CFU (or any range or value betweenabout 1×10³ and about 1×10¹¹) of viable bacteria. A dose of acomposition of the invention can be administered at three times a day,twice a day, once a day, every other day, two times a week, weekly,biweekly, or monthly. One, two, or more doses of a composition of theinvention can be administered per day for about 1 week, about 2 weeks,about 1 month, about 2 months, about 3 months, about a year or more.

Compositions of the invention can be used daily as part of an oral careregimen. Using compositions of the invention as part of a daily oralcare regimen allows a user to achieve and sustain a variety of desiredoral care benefits, including but not limited to white, tartar-freeteeth.

A kit of the invention can contain a one month, two month, three month,four month, five month, six month, or 12 month supply of a compositionof the invention. Such a kit can include instructions for use of theenclosed food item. A composition of the invention can be packagedindividually within the kit. Alternatively, a plurality of the packagedcompositions can be provided in a storage container or outer package orcarton. In some embodiments a kit can include an additional componentthat aids in product stability, for example a dessicant oroxygen-consuming material (for example, within individual packaging orwithin a container including multiple food items).

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

What is claimed is:
 1. A mucosal patch, comprising: a protective layerthat is permeable; and a film layer comprising a first surface coupledto the protective layer, wherein the film layer comprises a stabilizedbacterial formulation comprising an isolated, non-pathogenic, hydrogenperoxide bacterial species or strain and a genetically modifiedLDH-deficient bacterial strain.
 2. The mucosal patch of claim 1, whereinthe protective layer is dimensioned to fit within a human oral cavity.3. The mucosal patch of claim 1, wherein the stabilized bacterialformulation comprises lyophilized bacteria.
 4. The mucosal patch ofclaim 1, wherein the stabilized bacterial formulation comprisesencapsulated bacteria.
 5. The mucosal patch of claim 4, whereinencapsulated bacteria comprises a matrix compound selected from thegroup consisting of casein, starch, a chitosan, and an edible wax oroil.
 6. The mucosal patch of claim 5, wherein the edible wax or oil hasa melting point of greater than 35° C.
 7. The mucosal patch of claim 1,wherein the protective layer further comprises an adhesive.
 8. Themucosal patch of claim 1, further comprising a barrier layer coupled toa second surface of the film layer in a reversible fashion.
 9. Themucosal patch of claim 1, wherein the protective layer is porous.